Objectives To express a TAT-PBX1 fusion protein using a prokaryotic expression system and to explore potential effects of TAT-PBX1 in the proliferation and senescence of human hair follicle-derived mesenchymal stem cells. Results The TAT-PBX1 fusion was produced in inclusion bodies and heterogenously expressed in Rosetta (DE3) cells. Immunofluorescence staining showed that TAT-PBX1 fusion proteins were internalized by human hair follicle-derived mesenchymal stem cells. The growth rate of cells was increased after treatment with more than 5.0 mu g/mL of TAT-PBX1. The rate of senescence-associated beta-galactosidase positive cells was reduced in the 10.0 mu g/mL TAT-PBX1 group (28%) than the 0 mu g/mL control group (60%). Cells treated with the TAT-PBX1 fusion protein showed higher expression of p-AKT (1.22-fold that of the control), which indicates that TAT-PBX1 activated AKT pathway after cellular uptake. Conclusions The TAT-PBX1 fusion protein increased the proliferation of hair follicle mesenchymal stem cells and delayed their senescence by activating the AKT pathway following internalization by cells.